TARDBP和HIF-1α在神经酰胺调控黑色素瘤糖酵解抑制增殖转移中的作用机制研究

In previous study, we have found that ceramide can influence the proliferation and metastasis characteristic of melanoma cell through the change of glycolysis level. The pilot experiment preliminary confirmed the regulating axis that "ceramide→TARDBP and HIF-1αgene→glycolysis→the proliferation and metastasis related biological behaviour of melanoma cell". In the formal project, we are going to screen the cell lines in which TARDBP is high-expression(or low-expression) and HIF-1αis high-expression(or low-expression).With these cell lines, we will construct the implantation tumor and metastatic tumor model of nude mouse. Then verify the suppose that the change of ceramide can alter the expression of TARDBP and HIF-1αgene resulting the change of melanoma's proliferation and metastasis phenotype through the regulation of glycolysis level. On these basics, on the one hand , we will discuss the question whether ceramide regulate glycolysis through the modulation of glycolysis substrate(product) concentration or the modulation of key enzyme of glycolysis. On the other hand, we will discuss which subtype of ceramide can mostly affect the glycolysis level and proliferation and metastasis phenotype of melanoma. At last, we will discuss the relationship between ceramide、TARDBP and HIF-1αgene、the expression of related molecules in glycolysis and the prognosis and condition of patient. Through these, we can provide science evidence for melanoma early diagnosis，therapy and early warning for prognosis, and construct a new model of study about translational medicine.

前期研究发现，神经酰胺能改变糖酵解水平影响黑色素瘤细胞增殖转移特性。预实验在黑色素瘤中初步证实"神经酰胺→TARDBP和HIF-1α基因→糖酵解→黑色素瘤增殖转移相关生物学行为"调控轴的存在。本课题将首先筛选TARDBP高表达和低表达、HIF-1α高表达和低表达细胞株，构建裸鼠种植瘤和移植瘤模型，从细胞和动物水平验证神经酰胺的改变影响了TARDBP和HIF-1α基因的表达，进一步调控糖酵解水平来影响了黑色素瘤的增殖转移表型；在此基础上，一方面探讨神经酰胺对糖酵解的调控是通过调节糖酵解底物（或产物）浓度变化还是糖酵解关键酶的改变，另一方面探讨何种亚型的神经酰胺对糖酵解水平及增殖转移表型的影响最大；最后在临床上，探讨神经酰胺、TARDBP和HIF-1α基因、糖酵解相关分子在肿瘤标本中的表达情况与患者病情及预后之间的相关性，为黑色素瘤早期诊断治疗和预后监测提供科学依据，形成转化医学研究新模式。

1. 免疫组化和Western bolt法对128例黑色素瘤患者组织进行检测，发现TARDBP（TAR DNA结合蛋白）TDP-43在肿瘤组织表达上调，高表达TDP-43黑色素瘤患者生存率显著降低。.2. 筛选TDP-43高表达黑色素瘤细胞系（A375和WM451），构建TDP-43 shRNA质粒并成功转染至细胞，采用细胞及动物模型证实敲除TDP-43显著抑制黑色素瘤细胞生长及转移能力。.3.Western blot法发现TDP-43与糖酵解关键因子葡萄糖转运体4（GLUT4）存在正性调控关系；抑制GLUT4表达可以显著抑制黑色素瘤细胞增殖活性、侵袭及转移能力，并下调黑色素瘤糖酵解水平。以上结果证实，TARDBP（TDP-43）主要调控GLUT4干扰糖酵解途径，进而发挥促进黑色素瘤细胞转移的作用。.4.神经酰胺合成酶6（CerS6）主要合成神经酰胺C16，首次证实CerS6在黑色素瘤细胞中低表达，筛选出WM35、WM451及SK-28三株黑色素瘤细胞系，构建CerS6过表达及沉默表达质粒，分别成功转染至三株细胞。结果显示过表达CerS6神经酰胺C16含量增加，负性调控HIF-1a后影响葡萄糖转运体1（GLUT1）和单羧酸转运蛋白转运体1（MCT1）下调糖酵解水平，并抑制细胞生长与转移；沉默CerS6则出现相反的现象。 .5. 进一步对沉默CerS6表达的三株黑色素瘤细胞系（WM35、WM451及SK-28）进行全mRNA芯片筛查，发现葡萄糖转运体1（GLUT1）上调和WNT5A下调。以上结果证实CerS6-神经酰胺-HIF-1a/GLUT1/WNT5A-糖酵解调控轴在抑制黑色素瘤转移过程起到十分关键的作用，可能为黑色素瘤的早期检测及晚期治疗供新的靶点。.6. 发现miR-33a-5p抑制黑素瘤细胞增殖能力并抑制黑色素瘤细胞糖酵解，而HIF-1α 参与miR-33a-5p介导的黑色素瘤细胞糖酵解过程。